Project summary In the future, an important step in medical treatment will be the replacement of diseased or injured organs with engineered organs grown outside the body. However, one major impediment towards this goal is the ability for tissue engineers to grow complex and functional blood vessels that supply oxygen and nutrients to these externally grown organs. A missing piece of the puzzle is in knowing how differences in thickness of the blood vessel wall forms throughout the vasculature. For this reason, researchers must understand how large- diameter blood vessels (located close to the heart) form thick vessel walls composed of layers of vascular smooth muscle cells (vSMCs), and how small-diameter vessels (located far away from the heart) form thin or absent layers of vSMCs. These differences in vessel wall thickness are critical requirements for the formation of a functional vasculature, but it is unclear how wall thickness is regulated. Thus, the long-term objective of this proposal is to elucidate the mechanisms governing the formation of blood vessel wall thickness. From our previous studies, we determined that developing blood vessels form thick vessel walls based on extent of exposure to blood flow forces. Thus, high-flow vessels recruit and attach to more vSMCs than low-flow vessels. What remains unknown are the specific mechanisms explaining how the force of blood flow (hemodynamic force) regulates vSMC recruitment and attachment. Using the mouse embryonic model, a team of undergraduate students, master?s students and the principal investigator will explore two major mechanisms regarding how vessel wall thickness is attained. In aim 1, we will test the whether hemodynamic force regulates expression of several Semaphorin3 signaling proteins (Sema3F/G and Sema3A) to control vSMC recruitment to high-flow vessels. This aim will be investigated by disrupting these Sema3 proteins to determine if this impedes vSMC recruitment to the vasculature, and by rescuing the vSMC recruitment defects exhibited upon reduction of blood flow, by reintroducing the Sema3 protein gradients. In aim 2, we will test whether hemodynamic force regulates the adhesiveness of vessels to promote vSMC attachment to vessels. This aim will be investigated by determining whether reduction of blood flow reduces the ability for vSMCs to attach to vessels by attenuating expression of adhesive molecules, such as extracellular matrix genes (or inhibitors to extracellular matrix-degrading enzymes), and by upregulating expression of extracellular matrix-degrading genes (Matrix metalloproteinase [Mmp] inhibitors). In this aim, we will also determine if use of Mmp inhibitors will enhance extracellular matrix formation, and as a result enhance the adhesion of vessels to vSMCs. By the determining the mechanisms of vessel wall investment with vSMCs, this will allow researchers to identify an appropriate set of molecular tools that will be used to engineer functional blood vessels, as well as repair damaged blood vessels in adults. Further, these studies will help support the training of undergraduate and master?s students in biomedical research.